Evaluation of the viability of biological cells through cytotoxicity measurements is important for assessing the effect of drugs, environmental pollutants, temperature and ionic extremes, radiation and irradiation, other cells and biological modifiers and other potentially adverse factors on cells and tissues. Cell membrane integrity is commonly used to indicate cell viability. Loss of the protective cell membrane results in loss of cell structure, loss of critical intracellular contents, loss of essential ionic gradients and loss of electrical potential. The inevitable result of a major loss of membrane integrity is cell death.
While there is not an exact equivalence between an intact cell membrane and the term "viability" (technically defined as the ability of a cell to maintain its existence), it is common to refer to cells that have intact membranes as "viable" cells and cells where the membrane has been irreversibly disrupted by a cytotoxic reagent as "dead" cells. There is, of course an intermediate condition where a cell that retains its membrane is in the process of "dying". A dying cell is not actually viable in that it cannot be cultured or reproduce. Dying cells are nevertheless often counted as living by common screening tests that rely on cell membrane integrity.
A common feature of loss of membrane integrity is the formation of pores which permit the passage of low molecular weight molecules (MW&lt;2000 Daltons) in and out of the cytoplasm. This enhanced permeability has been the basis of many cell viability and cytotoxicity evaluations. The most common methods in use for cytotoxicity/viability measurements are .sup.51 Cr release, which uses a radioactive dye, and Trypan Blue exclusion, which uses a colored, non-fluorescent dye.
Fluorescent dyes can be detected with greater sensitivity than can colored dyes and do not have the disposal problems associated with the use of radioactive materials. The combination of different colored fluorescent probes to simultaneously detect live and dead cells is generally described by Haugland, HANDBOOK OF FLUORESCENT PROBES AND RESEARCH CHEMICALS, Set 24 (1989-91). Haugland suggests combining the use of a non-polar membrane permeant derivative that is converted to a polar fluorescent product inside live cells and a polar tracer of another color that is excluded from cells until the membrane is ruptured. Propidium iodide is mentioned as the most common fluorescent polar tracer complementary to those that are retained in live cells, although the reference also notes the potential usefulness of ethidium bromide and ethidium monoazide. Haugland and others have described the use of fluorescein, particularly carboxy fluorescein diacetate, with propidium iodide, for example for use with flow cytometry for sorting live and dead cells.
Two problems with the use of carboxy fluorescein diacetate are leakage from cells following hydrolysis and sensitivity to intracellular pH. Inside living cells, calcein AM is hydrolyzed to a fluorescent dye, calcein, that is well retained by cells, as described in the Haugland reference. The reference does not mention the simultaneous use of calcein AM in combination with any other dye used to detect dead cells. Calcein is less sensitive to intracellular pH than other fluorescein compounds, making it less likely to be quenched in intracellular environments.
Although ethidium homodimer is also described in the Haugland reference (Set 28), the use of ethidium homodimer as a viability stain for cells is not described. Ethidium homodimer has exceptionally high affinity for nucleic acids that is several orders of magnitude greater than ethidium bromide or propidium iodide. It is this high affinity that facilitates the simple, no wash, procedure of this invention. The use of propidium iodide results in unbound dye causing background fluorescence, which usually necessitates washing the cells after combination with the reagent. In contrast, the greater affinity of ethidium homodimer for nucleic acids results in virtually no background fluorescence, thus removing the requirement for washing cells and simplifying and accelerating the assay. The lack of background fluorescence and increased sensitivity related to the use of ethidium homodimer and calcein AM results in a two-color fluorescence assay that can detect fewer cells using a smaller sample. The combination of ethidium homodimer and calcein AM has not previously been suggested as or demonstrated to be suitable for a simultaneous or sequential two-color fluorescence assay of cell viability and cytotoxicity .